Screening novel small molecule drugs against human ion channels is of utmost importance in ensuring the safety and efficacy of 21st century therapeutics. Ligand and voltage-gated ion channels making up more than 15% of FDA-approved drug targets, Unwanted blockage or stimulation of ion channels by drugs has caused severe side effects and deaths. Several blockbuster drugs were pulled from the market because of these side effects. There are at least two reasons to be concerned with this problem. First, patients cannot be guaranteed their safety when taking new medicines. Second, not every person is sensitive to these side effects, so they are effectively being denied treatment when a drug is pulled.
Currently, screening ion channels for unwanted drug interactions is an expensive and painstaking process. Though methods based on fluorescent plate readers have accelerated the screening process, electrophysiology is still required to understand how drugs and channels interact. Patch clamp electrophysiology requires live cells and sophisticated instrumentation, and a new cell line must be created for each channel or mutation to be studied. Reconstitution of channel proteins into artificial membranes (lipid bilayers) can be performed, however the purification and reconstitution of membrane proteins into lipid bilayers is an expensive, laborious and difficult practice.
U.S. Pat. No. 8,268,627 discloses a membrane system, the droplet-interface bilayer, as an alternative technology to cell-based methods or planar bilayer methods for study of membrane protein behavior. Briefly, a replica cell membrane is created by joining two independently-formed lipid monolayers together. Two aqueous droplets containing lipid vesicles are submerged under an oily hydrocarbon, typically hexadecane. The lipid vesicles fuse at the oil/water boundary of each droplet to form a self-assembled lipid monolayer around each aqueous droplet. When the two droplets are brought into contact, the hexadecane is squeezed out from between the monolayers to create a droplet-interface bilayer (DIB). Membrane proteins present within one of the aqueous droplets insert into the bilayer. An Ag/AgCl electrode within each droplet enables the application of a voltage and the measurement of ionic current flowing through channels in the DIB.
In vitro transcription and translation (IVTT) is a cell-free approach for the synthesis of proteins from DNA templates. Many IVTT systems and in vitro transcription (IVT) products that synthesize proteins from messenger RNA are now commercially available from a number of vendors and are capable of producing integral membrane proteins such as ion channels.
The cost of producing ion channels via IVTT reactions is relatively high when used with planar bilayer systems, where the aqueous compartment volumes are 100-1000 μL. Droplet-interface bilayers have emerged as a system with greater potential for this application because the required volumes are much lower (e.g., 200 nL per droplet) and the bilayers have higher stability.
Bacterial and viral membrane proteins with both α-helical and β-barrel structures, such as staphylococcal α-hemolysin, the potassium channel Kcv from chlorella virus, and the potassium channel KcsA from Streptomyces lividans, have been expressed by IVTT systems derived from E. coli and then incorporated in DIBs.
In vitro expression of eukaryotic membrane proteins presents unique challenges. In eukaryotic organisms, synthesis of membrane proteins proceeds in a series of steps that ensure proper folding and orientation. Much of a cell's interior is occupied by a network of membranes called the endoplasmic reticulum (ER). The ER contains a highly-specialized docking site capable of threading secretory proteins or inserting transmembrane protein segments into the lipid membrane. Called the translocon, this complex is believed to guide the channel's insertion such that only one orientation is possible. The insertion of membrane proteins into the ER is coupled to the protein's synthesis at the ribosome. When the first segment of protein emerges from the ribosome, it is bound by another protein complex called the signal recognition particle (SRP). The SRP binds to both the nascent peptide chain and the ribosome, thereby pausing protein synthesis. The ER contains a membrane-bound SRP receptor, which binds the SRP-peptide-ribosome complex. This membrane association effectively docks the ribosome with the translocon. When this occurs, the SRP is released and protein synthesis resumes, with each transmembrane domain threading into the membrane one at a time. For membrane proteins, like ion channels, the first transmembrane α-helix is the binding site for SRP. In short, the ribosome, SRP, SRP receptor, and translocon work together to insert and orient membrane proteins such as ion channels.
In many cases, bacterial IVTT systems are ill-suited for eukaryotic membrane protein expression within the DIB. Bacterial IVTT systems do not post-translationally modify the expressed proteins. More importantly, the translocon is absent from such bacterial systems. To be generally useful for animal or human membrane protein analysis, the IVTT-DIB approach would require all the components that eukaryotic organisms use for membrane protein expression.
To synthesize ion channels within a DIB system, an in vitro transcription/translation (IVTT) extract can be mixed with lipid vesicles and DNA for the desired membrane protein in the aqueous solution of one of the precursor droplets. Including an E. coli IVTT system in a DIB, membrane proteins such as the viral potassium channel protein (Kcv,) have been synthesized in situ. Unfortunately, these bilayer interfaces were found to be very unstable, lasting only a couple minutes. DIBs are even less stable when attempting to use any of a variety of eukaryotic expression systems, including rabbit reticulocyte lysate, wheat germ extract, and yeast extract.
There is a need in the art for new compositions and methods for screening ion channel-drug interactions that don't require cells or traditional patch clamping techniques. Further, there is a need in the art for improved cell-free methods to analyze eukaryotic membrane protein function.